Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals

Abstract

Antiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL.

The significance of autoantibodies against β2-glycoprotein I

“…54 Support for the identification of antiphospholipid antibodies as natural antibodies comes from the observation that healthy persons without APS can have memory B cells that produce antiphospholipid antibodies. 57 In several elegant studies, Fleming et al have shown that natural anti-␤ 2 GPI antibodies are involved in complement-mediated mesenteric ischemia/reperfusioninduced injury. 58,59 In addition, there are also indications that natural antiphospholipid antibodies are involved in acute graft rejection after renal transplantation.…”

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen‐driven affinity selection

“…According to the recent scientific literature, sequence analysis of antibody genes using Chang and Casali's method 10 is utilized most widely to ascertain the involvement of antigenic selection in disease pathogenesis in cases of different autoimmune disorders [12][13][14][15][16][17] and lymphomas. [18][19][20][21][22][23][24][25] However, Chang and Casali's method does not consider the differences in the intrinsic mutability of various regions of antibody genes.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…The baculovirus expression system has been used for basic research to analyze the molecular basis of specificity, 21,22,41,52 autoimmunity 32,33,36,39,41,42 or the function of intact antibodies or of isolated immunoglobulin domains. 18,40,[43][44][45][46] It is very easy and fast to generate recombinant viruses expressing H or L bearing specific mutations and to determine the impact of these mutations on the specificity or activity of a given antibody.…”

“…21,22,41 This strategy has been successfully used to analyze the catalytic activity of the 6D9 monoclonal antibody and the specificity of the PAC1 antibody that binds to the integrin αII b β3 on activated platelets. 18,52 Universal expression cassettes have also been designed either to directly clone the VH and VL domains of antibodies expressed by murine cells or by human B-cells isolated from patients 26,32,33,36,39 or to convert scFv or Fab fragments selected from phage-display libraries into an intact fully human or chimeric IgG. 29,34,35,37,38,42 The aim of many of these experiments was to isolate, characterize and validate antibodies for further therapeutic applications in the treatment of human diseases such as hemolytic disease of newborn, 31 cancer 23,27,[56][57][58] and infectious diseases, with the selection of neutralizing antibodies against hemorrhagic fever viruses (e.g., hantaan, Puumala viruses), 34,35,72 human cytomegalovirus, 37 influenza virus 38 or prion protein.…”

“…Several immunoglobulin isotypes from many different species, including murine IgG, 2,3,[21][22][23][24][25] chimeric IgG, [26][27][28][29][30] human IgG, [31][32][33][34][35][36][37][38][39] IgA, [40][41][42] IgM 41 and IgE, [43][44][45] as well as pig Igs 30,45 have been successfully expressed in insect cells. These molecules are all correctly processed (signal peptide cleaved), glycosylated and assembled (H2L2) with a production rate varying from 0.75 to 100 mg/l, depending on the antibody ( …”